Frequently Asked Questions (FAQ)

What is PICO?

Protein Interaction Coupling (PICO) is an ultra sensitive immunoassay for the detection and absolute quantification of proteins, protein interactions, and post-translational modifications. PICO combines the advantages of immunoassays with digital PCR technology, by translating protein status into DNA barcodes.

What can PICO measure?

With PICO you can measure single proteins, protein interactions, and post-translational modifications from any soluble biological sample. For a consistent nomenclature, we call them 'targets'.

What are the advantages of PICO compared to other protein detection assays?

PICO is designed to outperform existing protein detection and quantification solutions (e.g. western blot, co-immunoprecipitation (coIP), ELISA, or proximity ligation assay (PLA)) and to introduce powerful experimental possibilities:

• Absolute quantitative: PICO enables the absolute quantification of protein targets without the need of an external standard. The PICO data provides precise molar concentration of the target protein, protein interaction, or post-translational modification.
• Ultra-high sensitivity: The limit of detection (LOD) of PICO is in the femtomolar range, meaning with high quality antibodies you can detect 100 target molecules per cell. Due to the ultra-high sensitivity, the target protein can be detected from as low as 1,000 cells. 
• Zero background: PICO doesn’t have any background reactions. During the dPCR step, the sample is compartmentalized to exclude the possibility of target-independent background reactions. Therefore, there is zero signal in case no target is present in the dPCR reaction.
• Low sample input: The PICO assay only requires 2 µl of sample as an input, which is significantly lower than the sample requirements for western blot, coIP, or ELISA. Thus, together with ultra-high sensitivity, you can save on precious biological material. 
• Multiplex assays: The dPCR instruments can often detect at least five fluorescent colors, meaning five different antibodies with different PICOglue Labels. Thus, PICO offers extensive flexibility in designing parallel assays.

What is a couplex?

A couplex is a target bound by two labeled antibodies and is the molecular detection unit of the PICO assay.

How much more sensitive is PICO compared to a western blot?

Comparing the detection of the breast cancer oncoprotein HER2 with western blot and PICO, revealed that PICO is approximately 200-fold more sensitive than a western blot.

How much lab work is required for the PICO assay?

The PICO assay itself is a two-day process. On the first day, the sample is lysed and combined with the labeled antibody mix for overnight incubation. The next day the sample is highly diluted, mixed with the dPCR Master Mix and the PICO Probes, and analyzed by dPCR. The total hands-on time is approximately ~1h 30 min.

What are the requirements/recommendations concerning antibodies?

The PICO assay works best with monoclonal antibodies since they are often better characterized and defined by the antibody producer and the batch-to-batch variability is minimal. As the antibodies need to bind concurrently, choosing monoclonal antibodies raised against different epitopes on the protein is critical.

How stable are the labeled antibodies?

The labeled antibodies are stable for at least 12 months at 4°C.

What type of samples can be used in a PICO assay?

Any biological sample such as, cell lysate, supernatant, FFPE samples, tissue, extracellular vesicles (EVs), blood serum, etc. can be analyzed with PICO. The samples must be liquefied, lysed, and homogenized, following the general laboratory instructions for a particular sample. In case of measuring cells (e.g. human cell lines) the lysis steps are included in the PICO Amplification Core Kit user manual.

How does data quantification work?

Data analysis is performed using Actome’s web-based AMULATOR software. First, the raw dPCR data is exported from your dPCR instrument and is uploaded to AMULATOR for data analysis and basic statistical evaluation.

Where can I purchase the PICO kits?

The PICO kits are available for purchase in Actome’s webshop.

Don't hesitate to contact us if you have more questions!